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Abstract

Introduction

Primordial germ cells (PGCs) are the major population of cells in the developing bilateral
embryonic gonads. Little is known about the cellular responses of PGCs after treatment
with toxic chemicals such as busulfan during embryo development. In this study, we
investigated the elimination, restorative ability, and cell cycle status of endogenous
chicken PGCs after busulfan treatment.

Methods

Busulfan was emulsified in sesame oil by a dispersion-emulsifying system and injected
into the chick blastoderm (embryonic stage X). Subsequently, we conducted flow cytometry
analysis to evaluate changes in the PGC population and cell cycle status, and immunohistochemistry
to examine the germ cell proliferation.

Results

Results of flow cytometry and immunohistochemistry analyses after busulfan treatment
showed that the proportion of male PGCs at embryonic day 9 and female PGCs at embryonic
day 7 were increased by approximately 60% when compared with embryonic day 5.5. This
result suggests the existence of a compensatory mechanism in PGCs in response to the
cytotoxic effects of busulfan. Results of cell cycling analysis showed that the germ
cells in the G0/G1 phase were significantly decreased, while S/G2/M-phase germ cells were significantly increased in the treatment group compared with
the untreated control group in both 9-day-old male and female embryos. In addition,
in the proliferation analysis with 5-ethynyl-2′-deoxyuridine (EdU) incorporation,
we found that the proportion of EdU-positive cells among VASA homolog-positive cells
in the 9-day embryonic gonads of the busulfan-treated group was significantly higher
than in the control group.

Conclusions

We conclude that PGCs enter a restoration pathway by promoting their cell cycle after
experiencing a cytotoxic effect.

Introduction

The continuous maintenance of future generations in living organisms is preserved
by germ cell development. Thus, germ cell research is important to advance infertility
treatments and perform developmental studies. Elimination of endogenous germ cells
has been widely used in germ cell transplantation studies (for clinical purposes)
and germline chimera production (for research purposes). Several methods, including
gamma ray irradiation, X-ray irradiation [1-3], and busulfan administration [4-6], have been developed to eliminate endogenous germ cells in different vertebrate species.
These methods primarily induce DNA damage in target cells, resulting in loss of all
cellular mechanisms and ultimately cell destruction. Busulfan is an alkylating agent
that can induce target cell apoptosis when administered to cells or tissues. Until
recently, busulfan treatment was the preferred method of eliminating germ cells. Although
busulfan administration can induce side effects including lethality, sterility and
teratogenicity [7], the majority of studies have applied busulfan to eliminate germ cells in mouse and
rat testis because of its relatively higher cytotoxicity to target cells. After busulfan
administration, testicular germ cells undergo apoptosis; however, small populations
of spermatogonial stem cells survive in mice [8]. These surviving spermatogonial stem cells may be involved in restoration of the
germ cell population after reduction or withdrawal of busulfan toxicity [9].

Primordial germ cells (PGCs) are the precursors of germ cells in most vertebrates
and play an important role in early embryonic germ cells [10]. Elimination of PGCs by busulfan administration can be performed in early chicken
embryos because isolation and manipulation of PGCs from these embryos is simple compared
with other vertebrate embryos. In chickens, PGCs originate in the epiblast and migrate
through the hypoblast and blood to reach embryonic gonads. Busulfan administered to
chicken eggs at Eyal-Giladi and Kochav stage X [11] successfully eliminated all endogenous PGCs in the embryos. After busulfan treatment,
donor PGCs injected into the embryos migrated and colonized on the recipient gonads.
The proportion of donor-derived offspring was also increased significantly [5,12]. However, little is known about the cellular responses of PGCs after busulfan treatment.
In the present study, we conducted flow cytometric analysis to evaluate changes in
the PGC proportion and cell cycle status after busulfan treatment in chickens.

Methods

Experimental animal care

The care and experimental use of chickens were approved by the Institute of Laboratory
Animal Resources, Seoul National University (SNU-070823-5). White Leghorn chickens
were maintained according to a standard management program at the University Animal
Farm, Seoul National University, Korea. The procedures for animal management, reproduction,
and embryo manipulation adhered to the standard operating protocols of our laboratory.

Survival and hatching rates

To measure survival rates, egg candling was performed for each egg during the observation
period. Properly developing eggs were identified based on the clear demarcation of
light and dark side within the egg and the formation of a network of blood vessels
reaching toward the air space. Unfertilized eggs at day 3 were removed from the data
and hatching of the eggs occurred at approximately day 21.

Busulfan emulsification

Emulsification of busulfan and injection into chicken embryos was performed as described
by Nakamura and colleagues [5], with minor modifications. A schematic diagram of busulfan emulsification and injection
into eggs is shown in Figure 1. Approximately 40 mg busulfan (Sigma-Aldrich, St Louis, MO, USA) were dissolved in
1 ml N,N-dimethyl formamide (Merck, Darmstadt, Germany) and diluted 10-fold in phosphate-buffered
saline (PBS). For emulsification, an internal pressure micro kit (IMK-20; MCtech,
Siheung, Korea) was used as a dispersion-emulsifying system with a tube-shaped Shirasu
porous glass (SPG; pore diameter, 10 μm) membrane. The dispersed phase inside the
SPG membrane was filled with busulfan-solubilized solution, and the continuous phase
outside the SPG membrane was filled with the same volume of sesame oil (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA, USA) with 1% polyglycerol polyricinoleate (PGPR90;
Danisco, Denmark) (Figure 2). The internal pressure was injected using nitrogen gas while stirring the continuous
phase with a rotator. The final concentration of busulfan in the emulsion was 2 μg/μl
in sesame oil containing 1% PGPR90. To optimize the concentration of PGPR90, particle
size uniformity and the color of the emulsified solution with different concentrations
of PGPR90 were observed at time 0 and 1 day after emulsification. Newly laid White
Leghorn eggs at Eyal-Giladi and Kochav stage X were placed horizontally 1 hour before
injection. The optimal dose of busulfan was determined based on Nakamura and colleagues
[5]. A small hole was made at the sharp end of eggs to avoid air cell damage and 50 μl
busulfan emulsion (100 μg busulfan) were injected into the yolk under the blastoderm
through a small hole using a sharp needle. After injection, the hole was sealed and
the eggs were incubated at 37°C with 50 to 60% relative humidity until the gonads
were isolated at embryonic days 5.5, 7, 9 and 15.

5-Ethynyl-2′-deoxyuridine incorporation

To examine the proliferation activity of germ cells, approximately 10 μl of 10 mM
5-ethynyl-2′-deoxyuridine (EdU) in PBS was injected into the extra-embryonic blood
vessels 4 hours before embryonic day 9. After injection, the eggs were sealed with
Parafilm and incubated until the completion of embryonic day 9.

Immunohistochemistry

After collection of 5.5-day-old and 9-day-old embryos treated with busulfan at stage
X, the abdomen of the embryos was carefully dissected under a stereomicroscope and
the gonads were collected with sharp tweezers [13]. Whole gonads were then cryosectioned (thickness, 10 μm) or paraffin-sectioned (thickness,
6 μm) and stored for immunostaining. For the immunostaining analysis, gonadal sections
(after deparaffinization for paraffin-sectioned tissues) were washed three times with
PBS and blocked with blocking buffer, which was composed of PBS containing 5% goat
serum and 1% bovine serum albumin, for 1 hour at room temperature. Sections were then
incubated at 4°C overnight with rabbit anti-cVASA (chicken VASA homolog) antibody
to detect germ cells. After washing three times with PBS, sections were incubated
with secondary antibodies labeled with phycoerythrin (PE) or fluorescein isothiocyanate
(Santa Cruz Biotechnology) for 4 hours at room temperature. To detect incorporated
EdU, sections were further stained for Click-iT detection with Alex Fluor 594 (C10339;
Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Sections
were then mounted with Prolong Gold anti-fade reagent with 4',6-diamidino-2-phenylindole
(Invitrogen, Carlsbad, CA, USA) and visualized using fluorescence microscopy.

Statistical analysis

Statistical analysis was performed using Student’s t test in the SAS version 9.3 software (SAS Institute, Cary, NC, USA). The significance
of differences between control and treatment groups was analyzed using the general
linear model (PROC-GLM) in the SAS software. Differences between treatments were considered
significant at P <0.05.

Results

Emulsification conditions for busulfan with PGPR90 by IMK-20

For efficient emulsification of busulfan, PGPR90 was used as an emulsifier. The particle
size uniformity was observed under the microscope to confirm the effect of 0.0 to
10.0% PGPR90 on emulsification (Figure 2A). Emulsification did not occur with 0% PGPR90, whereas very low-level emulsification
was observed with 0.1% of PGPR90. With 1%, 5% and 10% PGPR90, the particle size uniformity
was maintained even after 24 hours (Figure 2B).

Survival and hatching rate of the chicken embryos after busulfan treatment

To evaluate teratogenic effects of busulfan treatment, we determined the survival
and hatching rates during embryonic development. The survival rates of the busulfan
treatment group were significantly lower than those of the untreated control group
during development. The survival rates of the control and busulfan-treated groups
showed no differences at day 3 but were significantly lower in the busulfan-treated
group after 7 days of incubation (P <0.05). Upon hatching, the survival rates of the two groups were significantly different
(P <0.01) (Table 1). Mean hatching rates of the untreated control and busulfan treatment groups were
84.47 ± 1.49% (n = 3, total events = 71) and 61.85 ± 2.59% (n = 3, total events = 144), respectively.

Elimination and restoration of PGCs after busulfan treatment

Depletion of PGCs after busulfan treatment was investigated by immunohistochemistry.
Whole gonads were collected at embryonic days 5.5 and 9 in both sexes and cryosectioned
prior to immunostaining. To identify germ cells, an anti-VASA primary antibody and
PE-conjugated secondary antibody were used. At day 5.5, numerous VASA-positive PGCs
were dispersed in the gonads of the male and female control group (Figure 3). However, the number of VASA-positive PGCs was greatly decreased in male and female
gonads of the busulfan-treated group. At day 9, VASA-positive germ cells were dispersed
throughout the male gonads and dispersed in the cortex region of female gonads. In
the busulfan-treated group, few VASA-positive germ cells were observed in male and
female gonads. Furthermore, the number of VASA-positive germ cells in busulfan-treated
female gonads at day 9 was slightly higher than that of busulfan-treated female gonads
at day 5.5 (Figure 3).

To examine the proportion of PGCs after busulfan treatment, VASA-positive cells in
the embryonic gonads were analyzed by flow cytometry. The mean proportions of PGCs
normalized to control PGCs in whole gonads at days 5.5, 7, 9 and 15 are shown in Figure 4. In day 5.5 embryonic gonads, the proportion of PGCs was decreased significantly
after busulfan treatment (male, 24%; female, 8%; normalized to control, n = 3). In day 7, 9 and 15 embryonic gonads, the proportion of PGCs was also decreased
significantly after busulfan treatment (male, 23%, 60% and 71%, respectively; female,
67%, 60% and 65%, respectively, normalized to control, n = 3). The rates of VASA-positive PGCs in all busulfan-treated groups regardless of
sex or developmental stage were significantly lower than those in the control groups
(P <0.001) (Figure 4). However, the proportion of PGCs in the busulfan treatment group was significantly
increased at embryonic day 9 in male embryos and at embryonic day 7 in female embryos
compared with embryonic day 5.5 (Figure 4). Consistent with this germ cell recovery phenomenon, chickens in the busulfan-treated
group produced functional sperms or eggs when they reached sexual maturity (n = 5 for male and n = 3 for female).

Figure 4.Proportion of primordial germ cells (PGCs) in the embryonic gonads after busulfan
treatment at stage X. (A) Representative flow cytometry dot plots of day 5.5, day 7, day 9 and day 15 embryonic
gonadal cells labeled with anti-VASA. (B) Proportion of PGCs in the embryonic gonads of the busulfan-treated group. Data were
normalized to the proportion of PGCs in the control. Bars indicate standard error
of the mean of triplicate analyses. ***P <0.001, significant difference compared with control.

Cell cycle regulation after busulfan treatment

To examine changes in the cell cycle of PGCs during the recovery period after busulfan
treatment, the cell cycle in VASA-positive PGCs of day 9 gonads was evaluated by flow
cytometry using propidium iodide staining. Representative and replicate cell cycle
results in the PGCs of day 9 gonads after busulfan treatment are shown in Figure 5A and 5B, respectively. In both males and females, the proportion of PGCs in the quiescent
phase (G0/G1) of the busulfan treatment group was significantly decreased compared with the control
group (male, 74.03 ± 0.68% to 68.65 ± 1.27%; female, 63.13 ± 1.03% to 58.17 ± 0.61%,
n = 3). In contrast, the proportion of PGCs in the proliferative phase (S/G2/M) of the busulfan-treated group was significantly increased compared with the control
group (male, 25.91 ± 0.68% to 31.35 ± 1.27%; female, 36.87 ± 1.03% to 41.83 ± 0.61%,
n = 3). The proportion of PGCs in the sub-G1 phase did not show significant changes between two groups in both males and females.

Proliferation of restored PGCs after busulfan treatment

To examine proliferation activity of the restored PGCs in the busulfan-treated group,
EdU-incorporated 9-day-old embryonic gonads were isolated and immunostained with anti-VASA
and EdU. Results showed that EdU-incorporated cell nuclei in the male and female gonads
of busulfan-treated groups were increased when compared with control groups (Figure 6A). Furthermore, we investigated the number of proliferating germ cells by counting
the number of EdU-positive cells among VASA-positive cells. The number of proliferating
germ cells increased by about 15% in busulfan-treated male gonads compared with the
control. Similarly, the number of proliferating cells increased by about 30% in busulfan-treated
female gonads (Figure 6B).

Discussion

To eliminate endogenous PGCs in chickens by busulfan treatment, a sustained-release
emulsion of busulfan using an SPG pumping connector was used in a previous study [5]. Here, we modified the emulsion methods using an internal pressure micro kit with
a tube-shaped SPG membrane and PGPR90. Using this method, we could simplify the preparation
of solubilized busulfan and obtain an increased hatching rate (61.85%) in the busulfan
treatment group when compared with previous studies that used the same busulfan dose
[5].

Busulfan, an alkylating agent, has been used for clinical studies of chronic myelogenous
leukemia and bone marrow transplantation [14,15]. Generally, busulfan targets slowly proliferating and nonproliferating cells. The
mechanism of action of busulfan has been identified as DNA alkylation leading to DNA–DNA
cross-linking [16], which causes cell death and/or cellular senescence through the ERK and p38 pathways.
Busulfan also functions as a mitogen-activated protein kinase [17]. Conservation of antimitotic pathway across various cell types remains unclear. Busulfan
can specifically target and kill germ cells in embryonic gonads or testes, leading
to the depletion of endogenous germ cells. PGCs, which are a precursor of gametes,
may therefore be a major target for the germ cell depletion and sterilization. To
target PGCs, busulfan should be administered at very early embryonic stages during
which PGCs are formed. There are about 30 PGCs in the blastoderm of a fertilized hen
egg [18]. Therefore, busulfan has been used to produce PGC-mediated germline chimeras by direct
injection into blastoderm of fertilized eggs in chickens [5,6,12]. When injected at Eyal-Giladi and Kochav stage X, busulfan efficiently removed endogenous
PGCs [11]. To our knowledge, restoration of endogenous PGCs after busulfan treatment has not
been reported to date.

In both sexes, the relative PGC ratios of the busulfan-treated group to the normal
embryos at 9 days were markedly higher than that those at day 5.5. Also, sexually
mature male and female chickens treated with busulfan at stage X were able to produce
functional sperms or eggs. These results indicated that germ cells were recovered
from the cytotoxic effects of busulfan during development. We thus hypothesized the
existence of a compensation mechanism to recover from busulfan toxicity in PGCs. To
confirm the increase in PGCs after busulfan treatment, we conducted flow cytometry
to enumerate the increase in PGC number. The number of PGCs in the busulfan treatment
group recovered to ~60% that of the control group. This suggested the existence of
compensation and/or recovery mechanism in response to cytotoxic damage in PGCs, which
is one of the characteristics of stem cells. A strong defensive mechanism against
cytotoxic damage has been demonstrated in various stem cells, including spermatogonial
stem cells [8] and embryonic stem cells [19]. To determine whether this compensation is caused by changes in the cell cycle, we
conducted flow cytometry with propidium iodide staining to discriminate nonproliferating
and proliferating PGCs after busulfan treatment. The decrease in the proportion of
G0/G1-phase PGCs and the increase in that of S/G2/M-phase PGCs after busulfan treatment indicated that the cell cycle status of some
PGC populations changed from quiescent (G0) to proliferative (S/G2/M) phases. This change in cell cycle status was further confirmed by the proliferation
assay with EdU incorporation. We found that the proportion of EdU-positive cells among
VASA-positive cells was significantly higher in the busulfan-treated group.

Our results could be interpreted in two ways: first, a subpopulation of PGCs with
stem cell characteristics proliferated, while the majority of PGCs underwent apoptosis
after busulfan treatment; or second, proliferation of existing PGCs after busulfan
treatment suggested that PGCs possess defensive mechanisms against cytotoxicity. Consistent
with the first interpretation, there exists a side population of PGCs in mice [20], which have a greater ability to develop into pluripotent stem cells [21]. In addition, side population cells that differentiated from PGCs were enriched in
spermatogonia of developing mice testes [22]. However, the relationship between subpopulations of PGCs and proliferating PGCs
after cytotoxic effects was not investigated and little is known about the existence
of side population cells in chicken germ cells. Consistent with the second interpretation,
conserved expression of several pluripotency-related genes [23,24] and microRNAs [25] were identified in PGCs. The potential of PGCs to transform into pluripotent embryonic
germ cells [13,26] indicates that PGCs maintain their undifferentiated state and stem cell attributes
in their genetic status. To understand the compensation and/or restoration mechanisms
of chicken PGCs, it is necessary to characterize the proliferating PGC subpopulation
in busulfan-treated chicken gonads.

Conclusions

Our data suggest that endogenous PGCs can recover from the cytotoxic effects of busulfan.
The cell cycle status of PGCs shifted to a lower proportion in the G0/G1 phase and a higher proportion in the S/G2/M phase after busulfan treatment, which indicates that the recovery of PGCs is strongly
associated with the cell cycle transition. Our data increase our understanding of
PGCs and provide an important basis for germ cell plantation studies.

Abbreviations

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

HCL participated in manuscript writing, data analysis and interpretation, collection
and assembly of data. SKK participated in collection and assembly of data, data analysis
and interpretation. S-BP, SWK and S-BC participated in collection and assembly of
data. KJP and HJL participated in data analysis and interpretation. TSP and DR participated
in manuscript writing. JYH participated in conception and design and final approval
of the manuscript. All authors read and approved the final manuscript for publication.

Acknowledgments

This work was supported by a grant from the Cooperative Research Program for Agricultural
Science and Technology Development (project number PJ008240012012) from the Rural
Development Administration and from Regional Subgenebank Support Program of Rural
Development Administration, Republic of Korea.